Genetic and molecular analysis of the osmotically regulatedproU operon of Salmonella typhimurium
Abstract
When there is an increase in the osmotic strength of the environment of an organism, osmotic stress is encountered. Osmotic stress causes a rapid efflux of water, producing cellular dehydration. Organisms respond to this situation by increasing the intracellular concentrations of certain compounds like proline and glycinebetaine. These compounds act as osmotic balancers serving to prevent cellular dehydration without having an inhibitory effect on intracellular processes. In the enteric bacterium Salmonella typhimurium the increase of these osmotic balancers in response to osmotic stress if mediated by the ProU transport system, which is encoded by genes of the proU operon. As an initial step in understanding the mechanism by which the proU operon is expressed, the proU operon was cloned from S. typhimurium and the nucleotide sequence was determined for the transcriptional control region and part of the first structural gene. The predicted amino acid sequence of the gene product had homology to other proteins that are inner membrane associated components of binding protein-dependent transport systems. Mapping of the proU messenger RNA revealed endpoints that are close enough to sequences weakly resembling consensus bacterial promoters. By deletion analysis, the sequence mediating osmoregulated expression of the proU operon was shown to be contained on a 260 base-pair fragment, 60 nucleotides upstream and 200 nucleotides downstream of the mRNA start point. Strains carrying lacZ expression vectors containing proU fragments with the promoter, but not the DNA between positions +30 and +200 directed a high level constitutive expression of $\beta$-galactosidase, owing to increased levels of mRNA synthesis. This suggests that there is a negative regulatory element for the proU operon, downstream of the promoter, within the first structural gene. It was ruled out that this element is an osmotic stress-dependent transcription terminator. Mutations were identified that resulted in constitutive expression of the proU operon, and each mutation was closely linked to the proU locus and cis-dominant over the wild-type allele. The failure to obtain either recessive or unlinked mutations suggests that transcriptional regulation of the operon is not under the negative control of a specific repressor protein that is dispensable for cell viability.
Degree
Ph.D.
Advisors
Csonka, Purdue University.
Subject Area
Molecular biology|Genetics|Microbiology
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